In this proposal we would like to understand the role of 3D cell shape in the phenotypic status of vascular smooth muscle cells (VSMC). We will be using a combination of numerical modeling of vascular smooth muscle cell network in different 3D shapes and high resolution microscopy in micropatterns for experimental validation. Studying the role of cell shape on the phenotypes of VSMC will impact our understanding of the origins of vascular diseases such as atherosclerosis and how cell shape might regulate phenotypic states in many cell types. Micropatterning VSMC into 3D shapes provides a unique opportunity to segregate the effect of cell shape from other factors such as cell to cell contacts and extracellular matrix that could conflate cell shape effects. To identify the mechanisms by which cell shape changes calcium signaling and gene expression in VSMC, we have three overlapping aims. 1) To conduct numerical simulations of VSMC . We will develop a multi-compartmental ODE model of receptor-ligand interactions and their downstream effectors using Virtual Cell or COMSOL. We will study muscarinic receptor pathway (M3R), TGF and PDGF signaling and their physiological responses including Ca2+ and transcription factor activities which are differentially impacted by cell shape based on our preliminary data 2) To conduct experiments on VSMC in 3D microfabricated surfaces. VSMC will be seeded to 3D microfabricated surfaces where they could mimic the organization of VSMC in a medial layer of aortic tissue. I will characterize the plasma membrane distribution of receptors, calcium signaling and transcription factor activities in ellipsoid and spherical shapes using live cell imaging, fluorescence correlation spectroscopy and super-resolution microscopy. 3) To Conduct mRNAseq experiments on VSMC in 3D microfabricated surfaces. For an unbiased view of the changes triggered by cell shape, we will profile gene expression by mRNAseq and confirm results using immunofluorescence of differentially expressed gene products. We will identify genes that play a selective role in physiology and test their effects by siRNA ablation experiments.